Cloning, expression and characterization of the gene encoding the enolase from fusobacterium nucleatum

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Cloning, expression and characterization of the gene encoding the enolase from fusobacterium nucleatum. / Yakarsonmez, S; Cayir, E; Mutlu, O; Nural, B; Sariyer, E; Topuzogullari, M; Milward, Michael; Cooper, Paul; Erdemir, A; Turgut-Balik, D.

In: Applied Biochemistry and Microbiology, Vol. 52, No. 1, 01.2016, p. 23-30.

Research output: Contribution to journalArticlepeer-review

Harvard

Yakarsonmez, S, Cayir, E, Mutlu, O, Nural, B, Sariyer, E, Topuzogullari, M, Milward, M, Cooper, P, Erdemir, A & Turgut-Balik, D 2016, 'Cloning, expression and characterization of the gene encoding the enolase from fusobacterium nucleatum', Applied Biochemistry and Microbiology, vol. 52, no. 1, pp. 23-30. https://doi.org/10.1134/S0003683816010142

APA

Yakarsonmez, S., Cayir, E., Mutlu, O., Nural, B., Sariyer, E., Topuzogullari, M., Milward, M., Cooper, P., Erdemir, A., & Turgut-Balik, D. (2016). Cloning, expression and characterization of the gene encoding the enolase from fusobacterium nucleatum. Applied Biochemistry and Microbiology, 52(1), 23-30. https://doi.org/10.1134/S0003683816010142

Vancouver

Author

Yakarsonmez, S ; Cayir, E ; Mutlu, O ; Nural, B ; Sariyer, E ; Topuzogullari, M ; Milward, Michael ; Cooper, Paul ; Erdemir, A ; Turgut-Balik, D. / Cloning, expression and characterization of the gene encoding the enolase from fusobacterium nucleatum. In: Applied Biochemistry and Microbiology. 2016 ; Vol. 52, No. 1. pp. 23-30.

Bibtex

@article{5205dc5507994360bc521241d8491146,
title = "Cloning, expression and characterization of the gene encoding the enolase from fusobacterium nucleatum",
abstract = "The gene encoding enolase from Fusobacterium nucleatum (FnENO) was cloned and analyzed for the first time. The gene comprises of 1302 nucleotide base pairs and encodes 433 amino acids. The gene sequence alignment demonstrated the presence of several distinct insertions and deletions, compared with the human enzyme. The gene for recombinant FnENO was inserted into the pLATE 31 vector system and expressed in E. coli BL21(DE3) cells as a soluble protein. The protein was purified by affinity chromatography using a Ni-NTA agarose matrix and shown on SDS-PAGE to be a 46 kDa protein. The molecular weight of the octameric form of the purified recombinant protein was determined as being 375 kDa by size exclusion chromatography. Optimal enzyme activity was observed at pH 8.5 and the enzyme remained stable at a range of different temperatures from 30 to 60°C. Using 2-phosphoglyceric acid as substrate for the purified enzyme, KM, kcat and kcat/KM were determined as 0.48 mM, 20.4 s–1 and 4.22 × 104 M–1s–1, respectively. Potential drug binding sites of FnENO were detected using homology modeling. These data could facilitate the design of new inhibitors of F. nucleatum which has already been shown to be resistant to several known antibiotics.",
keywords = "Fusobacterium nucleatum, enolase, periodontal diseases, kinetic characterization, homology modeling",
author = "S Yakarsonmez and E Cayir and O Mutlu and B Nural and E Sariyer and M Topuzogullari and Michael Milward and Paul Cooper and A Erdemir and D Turgut-Balik",
year = "2016",
month = jan,
doi = "10.1134/S0003683816010142",
language = "English",
volume = "52",
pages = "23--30",
journal = "Applied Biochemistry and Microbiology",
issn = "0003-6838",
publisher = "MAIK Nauka/Interperiodica",
number = "1",

}

RIS

TY - JOUR

T1 - Cloning, expression and characterization of the gene encoding the enolase from fusobacterium nucleatum

AU - Yakarsonmez, S

AU - Cayir, E

AU - Mutlu, O

AU - Nural, B

AU - Sariyer, E

AU - Topuzogullari, M

AU - Milward, Michael

AU - Cooper, Paul

AU - Erdemir, A

AU - Turgut-Balik, D

PY - 2016/1

Y1 - 2016/1

N2 - The gene encoding enolase from Fusobacterium nucleatum (FnENO) was cloned and analyzed for the first time. The gene comprises of 1302 nucleotide base pairs and encodes 433 amino acids. The gene sequence alignment demonstrated the presence of several distinct insertions and deletions, compared with the human enzyme. The gene for recombinant FnENO was inserted into the pLATE 31 vector system and expressed in E. coli BL21(DE3) cells as a soluble protein. The protein was purified by affinity chromatography using a Ni-NTA agarose matrix and shown on SDS-PAGE to be a 46 kDa protein. The molecular weight of the octameric form of the purified recombinant protein was determined as being 375 kDa by size exclusion chromatography. Optimal enzyme activity was observed at pH 8.5 and the enzyme remained stable at a range of different temperatures from 30 to 60°C. Using 2-phosphoglyceric acid as substrate for the purified enzyme, KM, kcat and kcat/KM were determined as 0.48 mM, 20.4 s–1 and 4.22 × 104 M–1s–1, respectively. Potential drug binding sites of FnENO were detected using homology modeling. These data could facilitate the design of new inhibitors of F. nucleatum which has already been shown to be resistant to several known antibiotics.

AB - The gene encoding enolase from Fusobacterium nucleatum (FnENO) was cloned and analyzed for the first time. The gene comprises of 1302 nucleotide base pairs and encodes 433 amino acids. The gene sequence alignment demonstrated the presence of several distinct insertions and deletions, compared with the human enzyme. The gene for recombinant FnENO was inserted into the pLATE 31 vector system and expressed in E. coli BL21(DE3) cells as a soluble protein. The protein was purified by affinity chromatography using a Ni-NTA agarose matrix and shown on SDS-PAGE to be a 46 kDa protein. The molecular weight of the octameric form of the purified recombinant protein was determined as being 375 kDa by size exclusion chromatography. Optimal enzyme activity was observed at pH 8.5 and the enzyme remained stable at a range of different temperatures from 30 to 60°C. Using 2-phosphoglyceric acid as substrate for the purified enzyme, KM, kcat and kcat/KM were determined as 0.48 mM, 20.4 s–1 and 4.22 × 104 M–1s–1, respectively. Potential drug binding sites of FnENO were detected using homology modeling. These data could facilitate the design of new inhibitors of F. nucleatum which has already been shown to be resistant to several known antibiotics.

KW - Fusobacterium nucleatum

KW - enolase

KW - periodontal diseases

KW - kinetic characterization

KW - homology modeling

U2 - 10.1134/S0003683816010142

DO - 10.1134/S0003683816010142

M3 - Article

VL - 52

SP - 23

EP - 30

JO - Applied Biochemistry and Microbiology

JF - Applied Biochemistry and Microbiology

SN - 0003-6838

IS - 1

ER -